The long term objective of our research program is to define the basic defect in Cystic Fibrosis(CF). The approach described in this application will initially bypass the search for a specifid defect in CF. Instead, our specific aim is to identify a DNA marker closely linked to the disease locus. The study exploits the DNA sequence heterogeneity prevalent in the human population. Since these hereditary sequence polymorphisms are conveniently detected by using restriction endonucleases, the variable lengths of specifc DNA fragments generated from enzyme digestions have been used as markers in genetic analysis. By following the inheritance of a polymorphic DNA marker in families with CF patients, statistical methods are available to determine whether the disease is linked to the marker. A systematic screening of DNA markers distributed over the entire genome (excluding the sex chromosomes) should lead to the discovery of one such marker closely linked to the CF gene. This approach is expected to yield positive results because it is designed to examine the patient's genome directly rather than measuring parameters which could be due to secondary manifestation of the basic defect. Based on the size of the human genome and recombination frequencies, it has been estimated that approximately 150 to 400 markers mikght be required to map an unknown locus. However, this just sets the upper limit of the number of probes to be analyzed. In fact, a marker closely linked to Huntington Disease was found after screening only 12 random markers. To pursue our study, two-generation families with two or more CF children have been chosen as panels for screening DNA markers. DNA samples are prepared from lymphoblast cell lines established from each family member, digested with appropriate restriction enzymes, size-fractionated by agarose gel electrophoresis, transferred to DNA-binding membranes and hybridized with radioactively labelled DNA probes. The restriction fragment length polymorphisms are then revelaed by autoradiography. To facilitate analysis and storage of data we have developed computer-assisted systems. The discovery of a CF-linked DNA marker will likely provide an opportunity for carrier detection and prenatal diagnosis. More importantly, however, the marker should eventually permit identification of the CF gene. Identification of the basic biochemical defect in CF is a prerequisite for the development of effective therapy for this disorder.